We propose to develop a nanofluidic platform for the high-throughput restriction mapping of complete genomes. This platform consists of monolithically integrated fluidics for the extraction of chromosomal DNA from cells, its digestion by restriction endonucleases in a long nanochannel, and the high-resolution sizing of ordered restriction fragments. In contrast to other nanochannel-based approaches, we do not rely on the imaging of highly confined and elongated DNA molecules. Rather, fluorescently labeled DNA is digested by restriction endonucleases in a relatively large diameter (300 - 500 nm) channel in which nanochannel confinement and electrostatic forces prevent diffusive mixing of adjacent fragments. After digestion, the ordered fragments are electrophoretically driven to an injection point where the channel diameter decreases to ~100 nm. As each fragment reaches this point, it is accelerated by the higher electric field, creating separation from its trailing neighbors. Thse separated fragments migrate through a focused laser spot and the duration and integrated intensity of each fluorescence pulse is detected and analyzed to determine fragment size. We anticipate several advantages to our approach; it relies on nanochannels greater than 100 nm, ensuring that, after prototyping, devices can be fabricated using low-cost, high-throughput methods. The ability to fabricate very long nanochannels with diameters of 300 - 500 nm enables us to confine long genomic DNA in the reaction nanochannel, greatly reducing the need for map assembly from smaller DNA molecules. Integration of DNA extraction on chip increases the probability of interrogating intact chromosomal DNA, eliminating map assembly entirely and providing truly global coverage. Single-point detection obviates the need for image storage and analysis. In addition, many unique opportunities for pre- or post-mapping functionality are possible. We have conducted preliminary studies demonstrating the ordered injection of fragments from a reaction nanochannel into a smaller detection nanochannel and the ability of this approach to resolve neighboring fragments. Together with other elements developed by the Ramsey group and others, we believe that we would be generating restriction maps during the first half of the proposed project. Consequently, our team includes members with expertise in next generation sequencing (NGS) and bioinformatics. We will validate our restriction maps against reference sequences and physical maps generated using other methods. We will also demonstrate the utility of our restriction maps as scaffolds for de novo assembly of NGS data. Any errors or biases determined from these assessments will direct platform improvements.